Cloning, characterization and expression of the chitinase gene of Enterobacter sp. NRG4

Abstract

A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as antifungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purifi ed to near homogeneity using His-Tag affi nity chromatography. The purifi ed recombinant chitinase had a specifi c activity of 2041.6 U mg -1 . It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the K m, k cat and catalytic effi ciency (k cat /K m ) values of recombinant chitinase were found to be 1.27 mg ml -1 , 0.69 s -1 and 0.54 s -1 M -1 respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.

Transglycosylation by a chitinase from Enterobacter cloacae subsp. cloacae generates longer chitin oligosaccharides

“…G-1 18 , E. aerogenes 19 , and Enterobacter sp. NRG4 20 chitinases were characterized. ChiA was the first reported chitinase 21 from an Enterobacter sp and shared 26% sequence homology with Ec Chi1.…”

“…Wt (kDa) Optimum pH Optimum Temp (°C) TG activity Reference Enterobacter agglomerans ChiA 61 ND ND Not reported 21 Enterobacer sp.G-1 N-acetyl glucosaminidase 92 6.0 45 Not reported 18 ChiA 60 ND ND Not reported 61 E nterobacer sp. NRG4 ChiA 61 5.5 45 Not reported 20 E. aerogenes Chitinase 42.5 6.0 55 Not reported 19 E. cloacae subsp . cloacae Ec Chi1 42.5 5 40 Yes Present study ND – Not determined.…”

Production of N-Acetylglucosamine Using Recombinant Chitinolytic Enzymes

“…Protein concentrations were determined by bicinchoninic acid (BCA) method using bovine serum albumin (BSA) as a standard. Recombinant CHI was induced, overexpressed and purified by using optimized protocol [10].…”

“…The enzymatic breakdown of chitin by chitinases has been reported from our laboratory [9,10] as well as from others [11][12][13][14][15]. The chitinase gene (chi) of Enterobacter sp.…”

“…The chitinase gene (chi) of Enterobacter sp. NRG4 has already been cloned and characterized in our laboratory [10]. This recombinant CHI hydrolyzed swollen chitin into chitobiose (major fraction) and NAG (minor fraction).…”